Mol. Hum. Reprod. Advance Access published online on February 2, 2007
Molecular Human Reproduction, doi:10.1093/molehr/gal118
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Associations of MTHFR DNMT3b 4977 bp deletion in mtDNA and GSTM1 deletion, and aberrant CpG island hypermethylation of GSTM1 in non-obstructive infertility in Indian men
1 CSIRO Human Nutrition, Adelaide, Australia 2 Human Genetics Laboratory, Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, India
3 To whom correspondence should be addressed at: Genome Health and Nutrigenomics Laboratory, CSIRO Human Nutrition, Gate 13, Kintore Avenue, PO Box 10041, Adelaide, SA 5000, Australia. E-mail: varinderpal.dhillon{at}csiro.au
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Abstract |
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Methylenetetrahydrofolate (MTHFR) and DNMT3b play imperative roles in DNA synthesis and de novo methylation. GSTM1 is involved in detoxification of carcinogens. Mitochondrial DNA deletion has been associated with lower motility in human sperm. We analysed if polymorphisms in MTHFR (C677T and A1298C) and DNMT3b (C46359T) are associated with non-obstructive male infertility. We also analysed if folate, vitamin B12, homocysteine (Hcy), 8'-hydroxy-2'-deoxygnanosine (8-OHdG) levels, dietary folate intake and mtDNA deletion (4977 bp) affects fertility, such interactions are modified by deletion and methylation of GSTM1. In this case–control study, we included 179 oligoasthenoteratozoospermia patients and 200 fertile men. Single-nucleotide polymorphism analysis was performed by PCR-restriction fragment length polymorphism. The MTHFR (C677T and A1298C) and DNMT3b (C46359T) frequencies did not differ significantly in two groups. GSTM1 in association with mtDNA 4977 deletion is significantly associated with infertility. Plasma folate and vitamin B12 levels are decreased and total Hcy is elevated in infertile men. GSTM1 methylation status was investigated by methylation-specific PCR. Methylation is significantly correlated with GSTM1 reduced/loss of expression in infertile men. Infertile men have significantly higher 8-OHdG levels. Dietary folate intake is not linked with GSTM1 methylation. Low folate intake in association with CT + TT genotypes (C677T) has significant protective effect on GSTM1 methylation. Results indicate that micronutrients, 8-OHdG levels, mtDNA deletion and GSTM1 promoter methylation are frequent alterations in infertility.
Key Words: CpG island hypermethylation/DNMT3b/GSTM1/infertility/mtDNA/MTHFR
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Introduction |
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Male infertility in humans has been acknowledged as the cause of couple's inability to bear children in 20–50% of total cases. Genetic causes such as Y chromosome microdeletions, translocation and chromosomal aberrations have been identified as risk factors for male infertility (Foresta et al., 2001; Dohle et al., 2002).
Folate is central to nucleotide synthesis and DNA methylation. Folate deficiency is known to occur frequently and related hyperhomocysteinaemia is considered a risk factor for many diseases, including infertility. Several nutrients and their metabolites can influence gene expression. DNA methylation (both maintenance and de novo mC) influences chromatin structure and gene expression. If transcription of tumour suppressor or vital morphogenic protein is repressed, it may contribute to oncogenesis and abnormal embryogenesis. Primary or secondary folate deficiency can result in aberrant DNA methylation. Folate deficiency (DNA lesions owing to low dNTP levels) can contribute to pathologies of various diseases (Rampersaud et al., 2000, Figure 1).
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MTHFR is a key regulatory enzyme involved in folate metabolism, DNA synthesis and remethylation reactions. Homocysteine is a metabolic product of remethylation and trans-sulphuration reactions involving methionine. Total homocysteine (tHcy) levels could affect DNA synthesis and methylation. A common polymorphism in the MTHFR gene (C677T) results in a thermolabile phenotype associated with high levels of homocysteine. MTHFR gene mutation C677T could influence its biochemical activity and studies have shown that TT (mutant) has 30% activity when compared with CC. Similarly, A1298C polymorphism also reduces enzyme activity but to a lesser degree than C677T (Frosst et al., 1995; van der Put et al., 1998). Its deficiency could alter the synthesis of 5-methyl-tetrahydrofolate (5-MTHF), interrupt Hcy remethylation to methionine and cause hyperhomocysteinaemia. Folate deficiency-linked hyperhomocysteinaemia is a risk factor for many diseases including infertility. Recent studies have reported diverse associations between MTHFR C677T polymorphism and infertility (Bezold et al., 2001; Ebisch et al., 2003; Stuppia et al., 2003; Singh et al., 2005; Paracchini et al., 2006). Low folate coupled with MTHFR SNPs can alter RNA/DNA synthesis and has the potential to be linked with infertility (Stern et al., 2000). Animal model studies suggest, MTHFR plays a critical role in spermatogenesis due to exceptionally higher activity in adult testis than other organs (Chen et al., 2001).
Folate is essential in converting methionine to S-adenosyl methionine, the principle methyl donor in methylation. Adequate folate levels are vital for regulation of genomic methylation. In mammals at least three DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) are needed to establish genomic methylation during embryogenesis. DNMT1 maintains methylation patterns during replication. DNMT3a and DNMT3b act as de novo methyltransferases to establish methylation (Okano et al., 1999). Mutations in DNMT3b can lead to methylation including CpG island hypomethylation. DNMT3b C > T polymorphism in promoter region results in increased activity and has recently been identified as a risk factor for many diseases.
DNA is prone to oxidative stress and a high level of oxidative damage in sperm DNA may be involved in male infertility. Human semen contains substantial amounts of glutathione-S-transferase (GST) which can satiate the toxicity of reactive oxygen species (ROS) to sperm (Mukhtar et al., 1978). The GST gene family produces isoenzymes that play an important role in protecting cells from oxidative stress (Mann et al., 2000). Reduced GST activity associated with increased ROS can cause sperm membrane damage (Gopalakrishnan and Shaha, 1998). GSTM1 with antioxidant properties is expressed in testis and is among the members of a family of enzymes involved in detoxification of carcinogens through diverse mechanisms (Autrup, 2000). It is involved in DNA synthesis and methylation pathway. It is polymorphic and half of the population is deficient due to a homozygous deletion. Higher DNA adducts have been reported in infertile men lacking GSTM1 (Paracchini et al., 2005). ROS can cause DNA damage by accumulating 8'-hydroxy-2'-deoxyguanosine (8-OHdG) in cells (Ames, 1998). Sperm DNA damage is associated with male infertility and 8-OHdG is a sensitive marker of ROS-mediated oxidative damage in sperm (Shen and Ong, 2000).
Human mtDNA codes for two rRNAs, 22 tRNAs and 13 polypeptides necessary for its respiration and oxidative phosphorylation. Less efficient mtDNA repair system accounts for 10–20-fold increased mutation rates when compared with genomic DNA. It has been implicated in some forms of infertility associated with spermatozoa motility. Previous studies have established low and poor sperm motility with higher incidences of 4977, 7345 and 7599 bp mtDNA deletion (Kao et al., 1995, 1998).
In this case–control study, we tested the hypothesis that GSTM1 epigenetic silencing is associated with infertility, dietary folate intake and SNPs in folate pathway genes (MTHFR C677T, A1298C and DNMT3b C46359T). We examined if folate, vitamin B12, Hcy and 8-OHdG levels are altered in male infertility and sperm mtDNA deletion affects fertility and such associations are modified by GSTM1 polymorphism.
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Materials and methods |
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Patients and tissue collection
Semen and testicular biopsy samples from 179 infertile male and 200 fertile men were snap frozen in liquid nitrogen immediately after resection and stored at – 70°C. All men were between 25 and 35 years of age. Male infertility was diagnosed on the basis of results from three successive semen analyses according to the published criteria [World Health Organization (WHO), 1992]. The present study comprises 179 oligoasthenoteratozoospermic (OAT) and 200 fertile men with normal chromosome constitution. Infertile men had an infertility history of at least 2 years with their spouses having confirmed normal gynaecological assessment. Folate, tHcy and vitamin B12 levels were determined as per standard techniques. Patients and controls were age-matched, non-smokers, non-alcoholic and had no previous family history of infertility. Dietary folate intake, logged author as meant? at least 5 years prior to study was estimated to investigate if there was any relationship between GSTM1 methylation. Levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), sexual hormone-binding globulin (SHBG), testosterone and estradiol were measured by automated fluorescence detection system. Both intra- and total-assay variation was below the level of 2.5% and 4.5%, respectively. Consent forms were signed by all participants and the study was approved by the University Human Ethics Committee.
Genomic DNA isolation and PCR-RFLP analysis
Semen specimens, obtained from infertile and fertile men were used in the present study. The spermatozoa were separated from seminal plasma by centrifugation and washed with phosphate-buffered saline. The sperm pellet was lysed in buffer (30 x 106–1.2 x 107spermatozoa/5 ml) containing 6 M guanidinium, 30 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, 0.25 mg/ml proteinase K and 0.3 M ß-mercaptoethanol, and incubated at 55°C for 4–6 h. Isopropyl alcohol (twice of lysate volume) was added directly to the lysate and the tube was gently inverted back and forth until the DNA fibres clumped together. In most cases, DNA mass (in the form of a ‘cotton ball’) was free from transparent gel matrix (incompletely lysed protein). In cases where the DNA pellet remained attached to the protein gel, the pellet was mechanically separated from the protein gel with the help of fine forceps and scissors. The recovered DNA was washed with 70% ethanol and dissolved in TE (Tris–EDTA) buffer.
The MTHFR 677 C > T variant was identified using PCR-RFLP method as described previously (Frosst et al., 1995). Briefly, PCR primers for amplification of the MTHFR mutation generate a 198-bp fragment. C to T substitution at codon 677 creates a HinfI recognition sequence. The digested PCR product yielded a single 198 bp fragment for wild-type C allele, heterozygote (CT) yielded three fragments as 198-, 175- and 23-bp and mutant T allele yielded two fragments of 175- and 23-bp. Similarly, MTHFR 1298 A > C polymorphism was identified as described previously (van der Put et al., 1998). PCR product (163 bp) was digested with MboII to yield 56-, 31-, 30-, 28- and 18-bp fragments for the wild-type and 84-, 31-, 30- and 18-bp fragments for the homozygous mutant genotype.
DNMT3b C > T polymorphism in the promoter region (C46359T) was performed as described previously (Shen et al., 2002). The 380-bp PCR product was digested overnight with AvrII (New England BioLabs Inc.) at 37°C. Digested products were separated on a 2.5% agarose (Sigma, St Louis, Mo, USA) gel containing ethidium bromide. The C allele has single 380-bp band and T allele has 207- and 173 bp fragments.
GSTM1 genotype was determined as described previously (Bell et al., 1993; Linhares et al., 2005). A 215 bp PCR product indicated the presence of GSTM1 genotype. Each reaction was repeated twice (three times if there was no PCR product). Restriction digestion with HaeII was carried out to differentiate between GSTM1*A and GSTM1*B as per method described elsewhere (Fryer et al., 1993). Presence of GSTM1*A is indicated by 112 bp product while 132 bp product indicates GSTM1*B.
mtDNA deletion in sperm DNA was determined as described previously (Kao et al., 1998). Presence of deletion was ascertained by primer-shift PCR. Representative examples are shown in Figure 2.
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Sodium bisulphite DNA sequencing
DNA methylation studies were carried out using bisulphite sequencing and methylation-specific PCR (MSP). Briefly, 1 µg DNA was denatured by NaOH (50 µl; final concentration, 0.2 M) for 10 min at 37°C. One microgram of salmon sperm DNA (Sigma) was added as carrier before modification. Freshly prepared hydroquinone (30 µl; 10 mM; Sigma) and 520 µl of sodium bisulphite (3 M; pH 5.0; Sigma) were mixed, and samples were incubated under mineral oil at 55°C for 16 h. DNA samples were desalted through Wizard columns (Promega, Madison, WI, USA) and desulphonated by NaOH (final concentration, 0.3 M) treatment for 5 min followed by ethanol precipitation. DNA was resuspended in water and used immediately or stored at – 20°C. Bisulphite-modified DNA of 50–100 µl was used for bisulphite sequencing and MSP. Bisulphite-modified DNA is amplified using primers (F, 5'-GAGTTTTAGGGTTGGGTAGGG-3' and R, 5'-ACTACAC TCAATAAAACTTCCTCCC-3'). PCR products were sub-cloned in pCR2.1-TOPO vector by TA Cloning kit (InVitrogen). To determine the methylation status of 5' CpG island, four clones (each plate) were sequenced using an ABI PRISM Dye Deoxyterminator Cycle Sequencing kit and analysed on ABI PRISM 377 DNA Sequencer (ABI).
MSP for GSTM1 gene
DNA methylation in CpG island within the promoter regions was carried out by MSP. Primers used were: (Methylated) F, 5'-GAAGTTGGCGAGGTCGAGTT TC-3'; R, 5'-ACCCGCCACAACCCGAAAAAACG-3'; (Unmethylated) F, 5-GGGAAGTTGGTGAGGTTGAGTTTT-3' and R, 5'-CAACCCACCACAA CCCAAAAAACA-3'. Another primer pair was used to confirm these results: (methylated) F, 5'-TTTTGGGCGTTTTGATTTC-3' and R, 5'-CGAAACCTT ACTACGACCCC-3', and (unmethylated) F, 5'-TTTTTGGGTGTTTT GATTTT-3' and R, 5'-ACAAAACCTTACTACAACCC C-3'. For MSP, 50 ng of bisulphite-treated DNA was added to reaction buffer containing 1.25 mM deoxynucleotide triphosphate, 16.6 mM (NH4)2SO4, 67 mM Tris (pH 8.8), 6.7 mM MgCl2, 10 mM ß-mercaptoethanol, 0.1% DMSO, 10 pmol of forward and reverse primers specific to methylated and unmethylated sequences and 2.5 units of AmpliTaq Gold (PE Biosystems, Foster City, CA, USA). Conditions were 95°C for 5 min, followed by two cycles of 95°C for 30 s, 68°C for 45 s and 72°C for 45 s, two cycles at 66°C and 35 cycles at 65°C with a final extension cycle of 72°C for 5 min. Methylation- and unmethylation-specific PCR generated 157- and 161 bp products, respectively, for first pair and 154 bp products with the second set of primers. Internal negative (water blank) and positive controls were always included in the experiments. MSP negative samples were analysed twice. DNA isolated from normal peripheral lymphocytes from healthy individuals served as a negative methylation control. Universally methylated and unmethylated DNA (Chemicon, USA) was used as controls for MSP. MSP products were run on 3% agarose gel electrophoresis with ethidium bromide staining. A positive and negative control was included in each amplification reaction. Representative examples are shown in Figure 4E.
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RT–PCR for GSTM1
GSTM1 mRNA levels in testis from these patients were compared with expression in sperm from fertile men using semi-quantitative RT–PCR. Avian myeloblastosis virus reverse transcriptase (Promega, Mannheim, Germany) was used to synthesize cDNA and amplified in duplex reactions in a total volume of 25 µl containing 150 µM each dNTPs, 1.5 mM MgCl2, 10 pmol of each primer pair and 1.0 unit of Taq polymerase. After denaturation at 95°C for 5 min, 30 cycles of 30 s at 94°C, 30 s at 61°C and 1 min at 72°C were performed, followed by 10 min at 72°C. The following primers were used to amplify GSTM1: F, 5'-actttcccaatctgccctac-3'; and R, 5'-ttctggattgtagcagatca-3' (191 bp PCR product). GAPDH1 was used as internal control. Each reaction was performed in triplicate. PCR products were electrophoresed on 2% agarose gels and quantified using a densitometer (Molecular Dynamics). Fold changes in expression in testicular cells from infertile and fertile men were calculated. A semi-quantitative analysis of gene expression was performed in replicate in three independent experiments in each individual. A given gene was considered down-regulated in a tissue when mRNA level was less than two standard deviations. Representative RT–PCR results are shown in Figure 4F.
Measurement of 8-OHdG contents in testicular biopsy DNA and sperm
8-OHdG levels were measured by HPLC-ECD as described previously (Shigenaga et al., 1994) in infertile and fertile individuals. 8-OHdG and deoxyguanosine (dG) were used as standard controls and measurements are expressed as number of 8-OHdG molecules per 105 dG.
Statistical analysis
The observed genotype frequencies for fertile and infertile men were compared with each other using Hardy–Weinberg equation (observed allele frequencies) using Chi square test. Odds ratios (ORs), 95% confidence intervals (CIs) and specificity were calculated using a 2 x 2 contingency table and Fisher exact test. All statistical analyses were performed with Prism 4 software (GraphPad Software Inc., San Diego, CA, USA). GSTM1 genotype was classified as either null (homozygous deletion) or non-deleted (wild-type). All P-values were two-tailed and P < 0.05 was considered statistically significant.
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Results |
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MTHFR, DNMT3b, GSTM1 polymorphisms and 4977 bp deletion in mtDNA
We analysed SNPs in MTHFR (C677T and A1298C) and DNMT3b (C46359T) in 179 infertile and 200 fertile men using PCR-RFLP method. The results are summarized in Table IA. The genotype and allele frequencies of C677T and A1298C were not statistically different in fertile and infertile men, except when CT and TT (C677T) taken together, compared with homozygous genotype (OR: 1.54; P = 0.046; Table IA and B). GSTM1 allele B is significantly associated with infertility (OR: 1.87; P = 0.0281; Table IA). A/B genotype frequency is significantly higher in fertile men (OR: 3.41; P = 0.025; Table IA). We found tight linkage (MTHFR) as variant 677TT/1298CC, 677CT/1298CC and 677TT/1298AC were not observed in this study. Significantly higher haplotype frequencies of CCAA and CTAC (OR: 2.21; P = 0.0139) in infertile men; and CCAC (OR: 6.04; P < 0.0001), CCCC (OR: 6.81; P = 0.0015) and CTAA (OR: 2.21; P = 0.0139) in the fertile group were observed (Table II). Sperm mtDNA deletion in fertile and infertile men differed significantly (OR: 6.92; P < 0.0001; Table IA) and was not associated with GSTM1. It is significantly higher in infertile group irrespective of GSTM1 genotype (OR: 4.76; P = 0.0013; OR: 12.34; P < 0.0001; Table IIIA). Genotype combinations like CCWWAA and CTWWAC (OR: 2.42; P = 0.0243) were found significantly in infertile men while CCWWAC (OR: 6.16; P = 0.0017), CCNNAC (OR: 6.06; P = 0.0199), CCWWCC (OR: 15.55; P = 0.0011) and CTWWAA and CTNNAA were found in fertile men (Table IIIB). No significant variation in sperm number, motility, morphology and vitality was observed (data not shown). CT and TT genotypes have some protective effect on fertility.
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Micronutrients and polymorphism
Plasma folate levels were lower in infertile men when compared with fertile men (Table IV). tHcy levels in 1298AA/677TT were significantly higher in infertile men than fertile men (16.7 versus 9.2 µmol/l; P < 0.0001). Higher tHcy levels were observed in infertile men across all combinations with less significance level (Table IV). Similar trends were observed with B12 levels across various genotype combinations in fertile and infertile men (Table IV). It is plausible that plasma folate levels may modify the effect of germline MTHFR variants impacting on infertility risk. Low plasma folate levels are associated with higher infertility risk in analysis controlling for matching factors. Odds ratio comparing highest with lowest fertility was 2.51 (Figure 3).
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Hypermethylation and expression of GSTM1 gene and polymorphisms in MTHFR
GSTM1 promoter contains 144 bp long CpG island (> 70% GC content; CpG ratio = 0.76; Figure 4A). We detected no methylation in CpG island both in normal lymphocytes and fertile testicular biopsies. However, GSTM1 exhibited frequent but localized methylation in testicular biopsies from infertile men (Figure 4B and C). GSTM1 promoter hypermethylation was only significantly observed in 40 infertile men (Table V). We observed significant association of GSTM1 hypermethylation and C677T polymorphism. T allele frequency was significantly low in methylated-GSTM1 group than unmethylated-GSTM1 group (0.19 versus 0.39; P = 0.0104). The germline variant carrying the T allele (CT or TT) showed low frequency of aberrant GSTM1 methylation (OR = 0.29; P = 0.0034). The protective effect seemed stronger for CT (0.32; P = 0.0088) than TT (OR = 0.19; P = 0.0331). Methylation levels were lower in DNMT3b mutants than other genotypes (Figure 4D). Dietary folate intake is not significantly associated with GSTM1 methylation (OR: 1.19; Table VIA and B). We observed 677C allele to augment risk of GSTM1 methylation associated with low dietary intake and 677T allele has significant protective effect on GSTM1 methylation with low dietary folate intake. The individuals homozygous for 1298A allele also exhibited increased association of low folate with GSTM1 methylation, consistent with our expectations (Table VIB).
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GSTM1 expression was detected in normal tissues of fertile and infertile men showing unmethylated/hemi-methylated GSTM1. Forty testis samples of infertile men with methylation have significant reduction (complete loss/reduced expression) in GSTM1 expression. We found significant differences in GSTM1 expression between infertile (mean ± SE) and fertile testis (0.73 ± 0.07 versus 1.68 ± 0.07 AU; P < 0.0001). Its expression in infertile men showing methylation (0.06 ± 0.09 AU) was significantly lower when compared with hemi-methylation or unmethylation (1.05 ± 0.10 AU; P < 0.005). We concluded that GSTM1 methylation leads to its low expression.
Levels of 8-OHdG, GSTM1 genotype and 4977 bp deletion in mtDNA
Infertile individuals with a GSTM1 null-type genotype have significantly higher levels of 8-OHdG in sperm DNA than individuals with a wild-type genotype. This trend persisted when 8-OHdG levels were compared in these groups alongwith mtDNA 4977 deletion (Figure 5). No significant difference was observed in hormone levels of testosterone, FSH, LH, SHBG and estradiol in these men (Table VII). We did not find any significant differences with specific genotypes and/or methylation status (data not shown).
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Discussion |
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In the present study, we concomitantly evaluated the associations of common polymorphisms in MTHFR (C677T and A1298C) and DNMT3b (C46359T) gene, involved in folate metabolism and their associated risk for infertility. The prevalence of these genotypes were also evaluated in conjunction with GSTM1 genotype, 4977 bp deletion in mtDNA and micronutrients levels of folate, homocysteine and vitamin B12. This study showed that plasma folate level was associated with infertility risk. Our findings demonstrate relevance of folate metabolism in susceptibility to infertility among Indian male population. A few previous studies have evaluated the association of MTHFR C677T polymorphism in infertile patients from Germany, The Netherlands, Italy, India, South Korea and China (Bezold et al., 2001; Ebisch et al., 2003; Stuppia et al., 2003; Singh et al., 2005; Park et al., 2005; Lee et al., 2006; A et al., 2007). Five of them (Bezold et al., 2001; Singh et al., 2005; Park et al., 2005; Lee et al., 2006; A et al., 2006) have reported significant increase in the frequency of mutants and heterozygotes among infertile men. However, another report (Ebisch et al., 2007) concluded that this polymorphism is not a risk factor for male factor subfertility and emphasized the significance of folates in spermatogenesis. Here, we report similar findings regarding the association of MTHFR and infertility. Our results are in stark contrast to that of Singh et al. (2005) from India in which they showed the increased frequency of mutants and heterozygotes in infertile individuals. This difference may be explained on the basis of the subjects studied. The majority of patients in their study (Singh et al., 2005) belong to azoospermic category, whereas in our study all patients were OAT type. Similarly, an Italian population also showed no significant difference in the frequency of homozygotes and heterozygotes, and the authors suggested some gene(s) other than MTHFR in the DNA methylation pathway could affect the male infertility (Stuppia et al., 2003). Folate deficiency has been shown to reduce proliferation of various cell types (Zhu and Melera, 2001). It has been established that folate intake is very important for male infertility, and depending upon dietary habits, this level in human serum may differ in different countries. It has already been shown that sperm concentration is significantly increased by folic acid and zinc sulphate treatment from fertile and subfertile men with CC genotype (MTHFR) but not from CT and TT genoype (Ebisch et al., 2003).
It has been established that oxidative stress can cause DNA damage in human sperm and can cause infertility (Hideya et al., 1997). In this study we found significantly higher 8-OHdG levels in infertile men than fertile men, when analysed in relation to GSTM1 genotype and mtDNA 4977 bp deletion. These observations give further support to the notion that sperm 8-OHdG levels may act as a good marker (Chen et al., 2002) of predicting the presence of oxidative stress in testis and their ability to cause potential damage to sperm DNA and their ability to induce fertilization. Oxidative stress can cause folate depletion. There are two mechanisms that can explain the occurrence of oxidative folate cleavage. First, oxidation of pteridine ring leads to dihydrofolate and then folic acid thereby retaining folate as an active cofactor. Second, reduced folates are particularly prone to oxidative scission thus rendering the product metabolically inactive as a cofactor (Suh et al., 2001). This can lead to hyperhomocysteinaemia. High levels of homocysteine can lead to auto-oxidation through the production of hydrogen peroxide (H2O2)—a harmful reactive oxygen metabolite (Loscalzo, 1996). It is, therefore, anticipated that its increased production may be associated with Hcy-mediated DNA damage. Hcy increases intracellular H2O2 generation, inducing apoptosis as a result of severe DNA damage. Oxidative stress induces peroxidative damage in sperm's plasma membrane and damage in mitochondrial and nuclear DNA (Aitken and Krausz, 2001). There is evidence to suggest that H2O2 treatment causes more damage in asthenozoospermic infertile men than in normozoospermic infertile and fertile men (Hughes et al., 1996). Hyperhomocysteinaemia leading to precocious atherosclerosis of testicular arteries could be one mechanism behind MTHFR polymorphism and infertility. Absence of a functional copy of GSTM1 and increased Hcy levels together can cause sperm membrane damage and also induce apoptosis. Serum folate and vitamin B12 levels were also tested because of their strong association to homocysteine metabolism, in terms of a possible therapeutic role. Although serum folate and vitamin B12 levels were lower in infertile men when compared with fertile men, but these differences did not reach statistical significance. Yet, our data clearly show that hyperhomocysteinaemia is more frequent in infertile than fertile men and can be regarded as one of the factors associated with infertility.
Epigenetic alterations in DNA without concomitant changes in underlying genetic code are now known to occur frequently in various human diseases. Our data also unveil, for the first time, that promoter hypermethylation of GSTM1 is a common event occurring in the testis of infertile men, with 40 of 120 showing such epigenetic aberrations. In this study, GSTM1 was shown to be expressed in human normal testicular tissue. It is under-expressed (
2-fold drop of level in matched normal tissue) in 47% of infertile men. It is a frequent abnormality in infertile men and it may have a significant role in causing infertility. Our results are in line with a previous report (Jhaveri et al., 2001) suggesting the decreased expression of H-cadherin by decreased intracellular folate is associated with hypermethylation of CpG islands. It is anticipated that GSTM1 repression under folate-deficient conditions may act as an effector of infertile phenotype having low folate levels. We have shown for the first time that GSTM1 promoter methylation is associated with low dietary folate intake in infertile men. This may occur either as a direct or indirect consequence of altered folate intake. Folate is a major methyl donor for DNA methylation. It provides substrate for MTHFR to convert 5,10-methylene-tetrahydrofolate (5,10-MTHF) to 5-MTHF subsequently metabolized to methionine (Ross, 2003). Alteration in methylation maintenance may be causally associated with global hypomethylation of the genome. Our data supports the notion that infertility risk posed by decreased dietary folate intake might in part be attributable to enhanced concurrent promoter hypermethylation in GSTM1 gene.
Localized folate diminution could result in a transformational change in testicular tissue. To compensate for the high demands of folates required for cell proliferation, folate receptors might be up-regulated. Under such conditions, TT genotype, associated with greater enzyme sensitivity to reduced availability of 5-MTHF, would maintain the required supply of 5,10-MTHF for nucleotide synthesis. However, CC or CT genotype having delayed inhibition due to its greater stability of MTHFR enzyme complex at lower concentrations of 5-MTHF could result in a compromised supply of single carbon units for the methylation of dUMP to dTMP. This could lead to increased DNA damage owing to reduced availability of 5,10-MTHF for DNA synthesis. This could lead to uracil mis-incorporation and DNA strand breaks. Dysregulation of methylation maintenance may be casually associated with global DNA hypomethylation. Alternatively, infertility risk due to low dietary folate intake may in part be attributed to the enhanced occurrence of concurrent hypermethylation in the promoter region of specific genes including GSTM1 as seen in the present study. Genetic and environmental factors are likely to be important in determining CpG-island methylation levels. CpG island methylation in GSTM1 gene showed significantly less methylation in DNMT3b TT homozygotes when compared with CC/CT genotype. DNMT3b mediates de novo DNA methylation, and C/T transition has been shown to increase 30% in vitro transcriptional activity (Shen et al., 2002). However, lower methylation levels of GSTM1 gene observed in DNMT3b TT homozygotes are contrary to expectations and could influence global DNA methylation independently and/or hypermethylation of CpG islands in promoter regions of various genes including GSTM1 as shown here. From our results we can conclude that TT mutants (DNMT3b) show significantly less methylation when compared with CC. We have found that a correlation exists between GSTM1 methylation and CC genotype of MTHFR C677T, thus establishing the fact that MTHFR enzyme integrity promotes DNA methylation. Our results support the hypothesis that genetic factors affecting function of DNMT3b and MTHFR genes, and low folate intake in CC (MTHFR) individuals may trigger the tendency of acquiring aberrant CpG island methylation in GSTM1 gene in infertile men.
Sperm chromatin structure and DNA integrity are known to have a critical influence on the fertilization process. Infertile men are found to have a higher fraction of sperm with chromatin defects and DNA breaks than fertile controls. Oxidative stress is suggested as one of the factors responsible for DNA damage in ejaculated sperm. Morphologically, abnormal spermatozoa and leukocytes are the main source of excess ROS generation in semen. Mechanisms like defective sperm chromatin packaging, chaotic apoptosis and oxidative stress can cause sperm damage. Therefore, in light of this GSTM1 polymorphisms and methylation, and mtDNA deletions might be assessed to determine the impact of these factors on infertility. Errors in meiotic pairing and recombination have also been suggested to play a role in male infertility. An increase in sex chromosomal aneuploidy could be related to higher susceptibility of XY bivalent to flawed segregation patterns as a result of smaller homologous regions available for synapsis and recombination (i.e. pseudo-autosomal region). Infertility can arise due to abnormalities in terminal differentiation (post-recombination) of germ cells. These factors along with the various parameters studied here in detail should be investigated in infertile men attending the fertility clinics.
The findings from the present study indicate that decreased levels of folate, B12 and increased levels of tHcy and 8-OHdG are associated with increased infertility risk. Similarly, an epigenetic change in GSTM1 methylation leading to its silencing along with its loss/reduced expression further aggravates the infertility risk. The present study showed positive association between mtDNA deletion and infertility. However, we could not find significant association among MTHFR, DNMT3b and GSTM1 polymorphisms and infertility.
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References |
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Submitted on November 13, 2006; resubmitted on December 21, 2006; accepted on January 2, 2007.
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